Carcinogenic response and other histopathological alterations in mice exposed to cigarette smoke for varying time periods after birth.

In spite of the outstanding role of tobacco smoking in human carcinogenesis, it is difficult to reproduce its effects in experimental animals. Based on the knowledge that a variety of mechanisms account for a higher susceptibility to carcinogens early in life, we have developed a murine model in which mainstream cigarette smoke becomes convincingly carcinogenic. The standard model involves exposure to smoke for 4 months, starting after birth, followed by an additional 3-4 months in filtered air. We evaluated herein the time- and dose-dependent response, at 7.5 months of life, of Swiss H mice that had been exposed to smoke for either 1, 2 or 4 months after birth. A one-month exposure, corresponding to a period of intense alveolarization, was sufficient to induce most inflammatory, degenerative and preneoplastic pulmonary lesions, including emphysema and alveolar epithelial hyperplasia, blood vessel proliferation and hemangiomas, reflecting an early proangiogenic role of smoking, and microadenomas bearing ki-67-positive proliferating cells as well as urinary bladder epithelial hyperplasia. Two months of exposure were needed to induce pulmonary adenomas and urinary bladder papillomas in males only, which highlights a protective role of estrogens in urinary bladder carcinogenesis. Four months, which in humans would correspond to the postnatal period, puberty, adolescence and early adulthood, were needed to induce other lesions, including tubular epithelial hyperplasia of kidney, bronchial epithelial hyperplasia and especially pulmonary malignant tumors. These findings highlight the concept that preneoplastic and neoplastic lesions occurring in adulthood can be induced by exposure to smoke early in life.

Early and late effects of aspirin and naproxen on microRNAs in the lung and blood of mice, either unexposed or exposed to cigarette smoke.

We recently showed that nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the lung tumors induced by cigarette smoke, either mainstream (MCS) or environmental (ECS), in female mice. We used subsets of mice to analyze the expression of 1135 microRNAs in both lung and blood serum, as related to the whole-body exposure to smoke and/or oral administration of either aspirin or naproxen. In a first study, we evaluated early microRNA alterations in A/J mice exposed to ECS for 10 weeks, starting at birth, and/or treated with NSAIDs for 6 weeks, starting after weaning. At that time, when no histopathological change were apparent, ECS caused a considerable downregulation of pulmonary microRNAs affecting both adaptive mechanisms and disease-related pathways. Aspirin and naproxen modulated, with intergender differences, the expression of microRNAs having a variety of functions, also including regulation of cyclooxygenases and inflammation. In a second study, we evaluated late microRNA alterations in Swiss H mice exposed to MCS during the first 4 months of life and treated with NSAIDs after weaning until 7.5 months of life, when tumors were detected in mouse lung. Modulation of pulmonary microRNAs by the two NSAIDs was correlated with their ability to prevent preneoplastic lesions (microadenomas) and adenomas in the lung. In both studies, exposure to smoke and/or treatment with NSAIDs also modulated microRNA profiles in the blood serum. However, their levels were poorly correlated with those of pulmonary microRNAs, presumably because circulating microRNAs reflect the contributions from multiple organs and not only from lung.

Interactions between ethanol and cigarette smoke in a mouse lung carcinogenesis model.

Both ethanol and cigarette smoke are classified as human carcinogens. They can synergize, especially in tissues of the upper aerodigestive tract that are targeted by both agents. The main objective of the present study was to evaluate the individual and combined effects of ethanol and smoke in the respiratory tract, either following transplacental exposure and/or postnatal exposure. We designed two consecutive studies in mouse models by exposing Swiss H mice to oral ethanol and/or inhaled mainstream cigarette smoke for up to 4 months, at various prenatal and postnatal life stages. Clastogenic effects and histopathological alterations were evaluated after 4 and 8 months, respectively. Ethanol was per se devoid of clastogenic effects in mouse peripheral blood erythrocytes. However, especially in mice exposed both transplacentally throughout pregnancy and in the postnatal life, ethanol administration was associated not only with liver damage but also with pro-angiogenetic effects in the lung by stimulating the proliferation of blood vessels. In addition, these mice developed pulmonary emphysema, alveolar epithelial hyperplasias, microadenomas, and benign tumors. On the other hand, ethanol interfered in the lung carcinogenesis process resulting from the concomitant exposure of mice to smoke. In fact, ethanol significantly attenuated some smoke-related preneoplastic and neoplastic lesions in the respiratory tract, such as alveolar epithelial hyperplasia, microadenomas, and even malignant tumors. In addition, ethanol attenuated cigarette smoke clastogenicity. In conclusion, preclinical studies provide evidence that, in spite of its pulmonary toxicity, ethanol may mitigate some noxious effects of cigarette smoke in the respiratory tract.

Blood and lung microRNAs as biomarkers of pulmonary tumorigenesis in cigarette smoke-exposed mice.

Cigarette smoke (CS) is known to dysregulate microRNA expression profiles in the lungs of mice, rats, and humans, thereby modulating several pathways involved in lung carcinogenesis and other CS-related diseases. We designed a study aimed at evaluating (a) the expression of 1135 microRNAs in the lung of Swiss H mice exposed to mainstream CS during the first 4 months of life and thereafter kept in filtered air for an additional 3.5 months, (b) the relationship between lung microRNA profiles and histopathological alterations in the lung, (c) intergender differences in microRNA expression, and (d) the comparison with microRNA profiles in blood serum. CS caused multiple histopathological alterations in the lung, which were almost absent in sham-exposed mice. An extensive microRNA dysregulation was detected in the lung of CS-exposed mice. Modulation of microRNA profiles was specifically related to the histopathological picture, no effect being detected in lung fragments with non-neoplastic lung diseases (emphysema or alveolar epithelial hyperplasia), whereas a close association occurred with the presence and multiplicity of preneoplastic lesions (microadenomas) and benign lung tumors (adenomas). Three microRNAs regulating estrogen and HER2-dependent mechanisms were modulated in the lung of adenoma-bearing female mice. Blood microRNAs were also modulated in mice affected by early neoplastic lesions. However, there was a poor association between lung microRNAs and circulating microRNAs, which can be ascribed to an impaired release of mature microRNAs from the damaged lung. Studies in progress are evaluating the feasibility of analyzing blood microRNAs as a molecular tool for lung cancer secondary prevention.

Pharmacological Modulation of Lung Carcinogenesis in Smokers: Preclinical and Clinical Evidence.

Many drugs in common use possess pleiotropic properties that make them capable of interfering with carcinogenesis mechanisms. We discuss here the ability of pharmacological agents to mitigate the pulmonary carcinogenicity of mainstream cigarette smoke. The evaluated agents include anti-inflammatory drugs (budesonide, celecoxib, aspirin, naproxen, licofelone), antidiabetic drugs (metformin, pioglitazone), antineoplastic agents (lapatinib, bexarotene, vorinostat), and other drugs and supplements (phenethyl isothiocyanate, myo-inositol, N-acetylcysteine, ascorbic acid, berry extracts). These drugs have been evaluated in mouse models mimicking interventions either in current smokers or in ex-smokers, or in prenatal chemoprevention. They display a broad spectrum of activities by attenuating either smoke-induced preneoplastic lesions or benign tumors and/or malignant tumors. Together with epidemiological data, these findings provide useful information to predict the potential effects of pharmacological agents in smokers.

Selective inhibition by aspirin and naproxen of mainstream cigarette smoke-induced genotoxicity and lung tumors in female mice.

The role of nonsteroidal anti-inflammatory drugs (NSAIDs) in smoke-related lung carcinogenesis is still controversial. We have developed and validated a murine model for evaluating the tumorigenicity of mainstream cigarette smoke (MCS) and its modulation by chemopreventive agents. In the present study, the protective effects of the nonselective cyclooxygenase inhibitors aspirin and naproxen were investigated by using a total of 277 Swiss H neonatal mice of both genders. Groups of mice were exposed whole-body to MCS during the first 4 months of life, followed by an additional 3.5 months in filtered air in order to allow a better growth of tumors. Aspirin (1600 mg/kg diet) and naproxen (320 mg/kg diet) were given after weanling until the end of the experiment. After 4 months of exposure, MCS significantly enhanced the frequency of micronucleated normochromatic erythrocytes in the peripheral blood of mice, and naproxen prevented such systemic genotoxic damage in female mice. After 7.5 months, exposure of mice to MCS resulted in the formation of lung tumors, both benign and malignant, and in several other histopathological lesions detectable both in the respiratory tract and in the urinary tract. Aspirin and, even more sharply, naproxen significantly inhibited the formation of lung tumors in MCS-exposed mice, but this protective effect selectively occurred in female mice only. These results lend support to the views that estrogens are involved in smoke-related pulmonary carcinogenesis and that NSAIDs have antiestrogenic properties. The two NSAIDs proved to be safe and efficacious in the experimental model used.

Characterization of genomic changes in the cervical pre-cancerous lesions and tumors induced by different types of human papillomaviruses.

Cervical carcinoma is the second most common malignancy among women in both incidence and mortality. Although much is known about the etiology and treatment of cervical cancer, the role of genetic alterations in the multistep pathway of cervical tumorigenesis is largely unknown. The aim of this study was to characterize the genomic changes in the cervical pre-cancerous lesions and tumors, induced by different types of human papillomaviruses. In this research was used the BlueGnome CytoChip oligo 2 × 105 K microarray for whole-genome oligo-array CGH. Microarray CGH analysis of 40 specimens was carried out-12 specimens from patients with early-stage squamous cell carcinomas; 19 specimens from patients with mild to moderate dysplasia and 9 with severe dysplasia. First we performed microarray CGH analysis of five DNA pools which contained the DNA from homogeneous groups of patients. The results revealed presence of micro chromosomal aberrations in chromosome region 14q11.2. According to the genome database these aberrations represent polymorphisms. Microarray analysis of DNA from 9 separate carcinoma lesions revealed a total of 26 aberrations in 14 chromosomes of nine patients. Our results showed the advantages of high-resolution chips in the clinical diagnosis of patients with cancerous and precancerous lesions caused by viral infection with HPV, but also highlight the need for extensive population studies revealing the molecular nature and clinical significance of different CNVs and the creation of detailed maps of variations in the Bulgarian population. This would facilitate extremely precise interpretation of specific genomic imbalances in the clinical aspect.

Modulation by licofelone and celecoxib of experimentally induced cancer and preneoplastic lesions in mice exposed to cigarette smoke.

Chronic inflammation plays a crucial role in cigarette smoke-related carcinogenesis. Accordingly, anti-inflammatory agents, such as nonsteroidal anti-inflammatory drugs (NSAIDs), provide a rational strategy in cancer chemoprevention. We assayed celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, and licofelone, an inhibitor of COX-1, COX-2, and 5- lipoxygenase (5-LOX), for the ability to modulate carcinogenesis in neonatal mice exposed to mainstream cigarette smoke (MCS) for 4 months and thereafter kept in filtered air for 3.5 months. A preliminary toxicity study and a chemoprevention study involved the use of 591 Swiss H mice. Exposure to MCS caused a variety of pulmonary emphysema, alveolar and bronchial epithelial hyperplasias, proliferation of blood vessels, microadenomas, adenomas and malignant tumors, as well as kidney tubular and urinary bladder papillary epithelial hyperplasias. Celecoxib (1600 mg/kg diet) and even better licofelone (960 mg/kg diet) were able to significantly attenuate the MCS-induced alterations of inflammatory nature, including pulmonary emphysema, alveolar epithelial hyperplasias and microadenomas and urinary tract hyperplastic lesions when given to mice according to a protocol that mimics an intervention in current smokers. Moreover, celecoxib attenuated the yield of lung adenomas and both NSAIDs showed some involvement in lowering the progression to cancer in the lung. Celecoxib exhibited some protective effects even when given according to a protocol involving its administration after discontinuation of exposure to MCS. However, both agents and especially celecoxib showed some hepatotoxicity and affected survival and body weight gain of mice when administered to MCS-exposed mice in the long term.

HPV prevalence and type distribution in women with normal or abnormal Pap smear in Bulgaria.

Human papillomavirus (HPV) is a well-known pathogen for lower genital tract neoplasias, yet little is known regarding HPV prevalence in Bulgaria. The aim of this study was to investigate the prevalence of HPV DNA and to determine HPV types distribution among women with normal and abnormal cytology. Cervical smears with different cytological diagnoses were collected from 355 Bulgarian patients. The cohort of patients selected is the biggest ever studied in this country. Using the Roche Linear Array HPV Genotyping Test, papillomavirus DNA was found in 217 out of the 355 samples, 164 of which had only one and 53 had more than one HPV type. The distribution of the viruses tested in 355 samples was as follows: (i) the most common type was HPV 16, which was found in 61 samples; (ii) the next most frequent HPV type was HPV 33, found in 14 of the samples. A high prevalence of HPV infection was observed in this study. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, two HPV vaccines are included in the National immunization schedule in Bulgaria. Thus, new clinical studies will benefit from patient stratification by the presence or absence of HPV, and by designing separate clinical trials specifically for HPV associated cancers.

Assay of lapatinib in murine models of cigarette smoke carcinogenesis.

Lapatinib, a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), is prescribed for the treatment of patients with metastatic breast cancer overexpressing HER-2. Involvement of this drug in pulmonary carcinogenesis has been poorly investigated. We used murine models suitable to evaluate cigarette smoke-related molecular and histopathological alterations. A total of 481 Swiss H mice were used. The mice were exposed to mainstream cigarette smoke (MCS) during the first four months of life. After 10 weeks, MCS caused an elevation of bulky DNA adducts, oxidative DNA damage and an extensive downregulation of microRNAs in lung. After four months, an increase in micronucleus frequency was observed in peripheral blood erythrocytes. After 7.5 months, histopathological alterations were detected in the lung, also including benign tumors and malignant tumors, and in the urinary tract. A subchronic toxicity study assessed the non-toxic doses of lapatinib, administered daily with the diet after weaning. After 10 weeks, lapatinib significantly attenuated the MCS-related nucleotide changes and upregulated several low-intensity microRNAs in lung. The drug poorly affected the MCS systemic genotoxicity and had modest protective effects on MCS-induced preneoplastic lesions in lung and kidney, when administered under conditions that temporarily mimicked interventions either in current smokers or ex-smokers. On the other hand, it caused some toxicity to the liver. Thus, on the whole, lapatinib appears to have a low impact in the smoke-related lung carcinogenesis models used, especially in terms of tumorigenic response.

Modulation by metformin of molecular and histopathological alterations in the lung of cigarette smoke-exposed mice.

The anti-diabetic drug metformin is endowed with anti-cancer properties. Epidemiological and experimental studies, however, did not provide univocal results regarding its role in pulmonary carcinogenesis. We used Swiss H mice of both genders in order to detect early molecular alterations and tumors induced by mainstream cigarette smoke. Based on a subchronic toxicity study, oral metformin was used at a dose of 800 mg/kg diet, which is 3.2 times higher than the therapeutic dose in humans. Exposure of mice to smoke for 4 months, starting at birth, induced a systemic clastogenic damage, formation of DNA adducts, oxidative DNA damage, and extensive downregulation of microRNAs in lung after 10 weeks. Preneoplastic lesions were detectable after 7.5 months in both lung and urinary tract along with lung tumors, both benign and malignant. Modulation by metformin of 42 of 1281 pulmonary microRNAs in smoke-free mice highlighted a variety of mechanisms, including modulation of AMPK, stress response, inflammation, NFκB, Tlr9, Tgf, p53, cell cycle, apoptosis, antioxidant pathways, Ras, Myc, Dicer, angiogenesis, stem cell recruitment, and angiogenesis. In smoke-exposed mice, metformin considerably decreased DNA adduct levels and oxidative DNA damage, and normalized the expression of several microRNAs. It did not prevent smoke-induced lung tumors but inhibited preneoplastic lesions in both lung and kidney. In conclusion, metformin was able to protect the mouse lung from smoke-induced DNA and microRNA alterations and to inhibit preneoplastic lesions in lung and kidney but failed to prevent lung adenomas and malignant tumors induced by this complex mixture.

Transplacental clastogenic and epigenetic effects of gold nanoparticles in mice.

The broad application of nanotechnology in medicine, biology, and pharmacology is leading to a dramatic increase of the risk of direct contact of nanoproducts, among which gold nanoparticles (AuNP), with the human organism. The present study aimed at evaluating in vivo the genotoxicity of AuNPs with average size of 40 nm and 100 nm. A single intraperitoneal treatment of adult male and female Swiss mice (strain H) with AuNPs, at a dose of 3.3 mg/kg body weight, had no effect on the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in bone marrow. Conversely, the transplacental treatment with AuNP-100 nm, but not with AuNP-40 nm, applied intraperitoneally at a dose of 3.3 mg/kg to pregnant mice on days 10, 12, 14, and 17 of gestation, resulted in a significant increase in the frequency of MN PCEs in both liver and peripheral blood of mouse fetuses. In parallel, the same treatment with AuNP-100 nm, but not with AuNP-40 nm, produced significant changes in microRNA expression. In particular, out of 1281 mouse microRNAs analyzed, 28 were dys-regulated more than two-fold and to a statistically significant extent in fetus lung, and 5 were up-regulated in fetal liver. Let-7a and miR-183 were significantly up-regulated in both organs. The data presented herein demonstrate for the first time the transplacental size-dependent clastogenic and epigenetic effects of AuNPs in mouse fetus, thus highlighting new aspects concerning the putative genotoxicity of AuNPs during a vulnerable period of life.

Relationships between pulmonary micro-RNA and proteome profiles, systemic cytogenetic damage and lung tumors in cigarette smoke-exposed mice treated with chemopreventive agents.

Assessing the correlation between molecular endpoints and cancer induction or prevention aims at validating the use of intermediate biomarkers. We previously developed murine models that are suitable to detect both the carcinogenicity of mainstream cigarette smoke (MCS) and the induction of molecular alterations. In this study, we used 931 Swiss mice in two parallel experiments and in a preliminary toxicity study. The chemopreventive agents included vorinostat, myo-inositol, bexarotene, pioglitazone and a combination of bexarotene and pioglitazone. Pulmonary micro-RNAs and proteins were evaluated by microarray analyses at 10 weeks of age in male and female mice, either unexposed or exposed to MCS since birth, and either untreated or receiving each one of the five chemopreventive regimens with the diet after weaning. At 4 months of age, the frequency of micronucleated normochromatic erythrocytes was evaluated. At 7 months, the lungs were subjected to standard histopathological analysis. The results showed that exposure to MCS significantly downregulated the expression of 79 of 694 lung micro-RNAs (11.4%) and upregulated 66 of 1164 proteins (5.7%). Administration of chemopreventive agents modulated the baseline micro-RNA and proteome profiles and reversed several MCS-induced alterations, with some intergender differences. The stronger protective effects were produced by the combination of bexarotene and pioglitazone, which also inhibited the MCS-induced clastogenic damage and the yield of malignant tumors. Pioglitazone alone increased the yield of lung adenomas. Thus, micro-RNAs, proteins, cytogenetic damage and lung tumors were closely related. The molecular biomarkers contributed to evaluate both protective and adverse effects of chemopreventive agents and highlighted the mechanisms involved.

DNA damage in exfoliated cells and histopathological alterations in the urinary tract of mice exposed to cigarette smoke and treated with chemopreventive agents.

Cigarette smoke (CS) is convincingly carcinogenic in mice when exposure starts at birth. We investigated the induction and modulation of alterations in the kidney and urinary bladder of CS-exposed mice. A total of 484 strain H Swiss mice were either sham-exposed or exposed since birth to mainstream CS (MCS) for 4 months. Dietary agents, including myo-inositol, suberoylanilide hydroxamic acid, bexarotene, pioglitazone and a combination of bexarotene and pioglitazone, were administered after weaning. Comet analyses showed that, after 2 and 4 months, MCS causes DNA damage in exfoliated urothelial cells, which can be prevented by myo-inositol and the peroxisome proliferator-activated receptor-γ ligand pioglitazone. After 7 months, the 17.6% of MCS-exposed male mice exhibited lesions of the urinary tract versus the 6.1% of sham-exposed mice, which emphasizes the role of sex hormones in urinary tract carcinogenesis. Myo-inositol and the RXR-specific retinoid bexarotene did not affect these alterations. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (Vorinostat) increased the incidence of kidney epithelium hyperplasia. Pioglitazone significantly enhanced the incidence of kidney lesions as compared with mice exposed to MCS only, indicating possible adverse effects of this antidiabetic drug, which were lost upon combination with bexarotene according to a combined chemoprevention strategy. RXR is a heterodymeric partner for peroxisome proliferator-activated receptor-γ, thereby modulating the expression of multiple target genes. In conclusion, there is contrast between the ability of pioglitazone to inhibit DNA damage in exfoliated cells and the alterations induced in the urinary tract of MCS-exposed mice, suggesting the occurrence of non-genotoxic mechanisms for this drug.

Inhibition of lung tumor development by berry extracts in mice exposed to cigarette smoke.

Cigarette smoke (CS) and dietary factors play a major role in cancer epidemiology. At the same time, however, the diet is the richest source of anticancer agents. Berries possess a broad array of health protective properties and were found to attenuate the yield of tumors induced by individual carcinogens in the rodent digestive tract and mammary gland but failed to prevent lung tumors induced by typical CS components in mice. We exposed whole-body Swiss ICR mice to mainstream CS, starting at birth and continuing daily for 4 months. Aqueous extracts of black chokeberry and strawberry were given as the only source of drinking water, starting after weaning and continuing for 7 months, thus mimicking an intervention in current smokers. In the absence of berries, CS caused a loss of body weight, induced early cytogenetical damage in circulating erythrocytes and histopathological alterations in lung (emphysema, blood vessel proliferation, alveolar epithelial hyperplasia and adenomas), liver (parenchymal degeneration) and urinary bladder (epithelial hyperplasia). Both berry extracts inhibited the CS-related body weight loss, cytogenetical damage, liver degeneration, pulmonary emphysema and lung adenomas. Protective effects were more pronounced in female mice, which may be ascribed to modulation by berry components of the metabolism of estrogens implicated in lung carcinogenesis. Interestingly, both the carcinogen and the chemopreventive agents tested are complex mixtures that contain a multitude of components working through composite mechanisms.

Differential carcinogenicity of cigarette smoke in mice exposed either transplacentally, early in life or in adulthood.

Cigarette smoke (CS) plays a dominant role in the epidemiology of human cancer. However, it is difficult to reproduce its carcinogenicity in laboratory animals. Recently, we showed that CS becomes a potent carcinogen in mice when exposure starts soon after birth. In our study, we comparatively evaluated the carcinogenic response to mainstream CS in mice at different ages. Neonatal mice were exposed daily for 4 months to CS, starting within 12 hr after birth, and sacrificed at 8 months. Adult mice were exposed for the same time period (3-7 months) and sacrificed at 11 months. Other mice were exposed transplacentally or both transplacentally and early in life. A total of 351 neonatal mice and 80 adult Swiss H mice were used. With varying intensity depending on age, CS induced pulmonary emphysema, bronchial and alveolar epithelial hyperplasia, blood vessel proliferation and hemangiomas and microadenomas in lung as well as parenchymal degeneration of liver. Histopathological alterations of kidney were only observed in mice exposed to CS early in life. Lung adenomas and malignant tumors of various histopathological nature were detected in neonatally exposed mice but not in adults. Transplacental CS induced the formation of lung adenomas in the offspring 8 months after birth. Previous exposure during pregnancy attenuated CS-related alveolar epithelial hyperplasia induced after birth. In conclusion, the carcinogenic response to CS varies depending on the developmental stage. The early postnatal life and the prenatal life are particularly at risk for the later development of CS-related tumors.

Prevention of cigarette smoke-induced lung tumors in mice by budesonide, phenethyl isothiocyanate, and N-acetylcysteine.

Lung cancer is the most important cause of death among neoplastic diseases worldwide, and cigarette smoke (CS) is the major risk factor for cancer. Complementarily to avoidance of exposure to CS, chemoprevention will lower the risk of cancer in passive smokers, ex-smokers, and addicted current smokers who fail to quit smoking. Unfortunately, chemoprevention clinical trials have produced disappointing results to date and, until recently, a suitable animal model evaluating CS carcinogenicity was not available. We previously demonstrated that mainstream CS induces a potent carcinogenic response when exposure of mice starts at birth. In the present study, neonatal mice (strain H) were exposed to CS for 120 consecutive days, starting at birth. The chemopreventive agents budesonide (2.4 mg/kg diet), phenethyl isothiocyanate (PEITC, 1,000 mg/kg diet), and N-acetyl-L-cysteine (NAC, 1,000 mg/kg body weight) were administered orally according to various protocols. The experiment was stopped after 210 days. Exposure to CS resulted in a high incidence and multiplicity of benign lung tumors and in significant increases of malignant lung tumors and other histopathological alterations. All three chemopreventive agents, administered to current smokers after weaning, were quite effective in protecting both male and female mice from CS pulmonary carcinogenicity. When given to ex-smokers after withdrawal of exposure to CS, the protective capacity of budesonide was unchanged, while PEITC lost part of its cancer chemopreventive activity. In conclusion, the proposed experimental model provides convincing evidence that it is possible to prevent CS-induced lung cancer by means of dietary and pharmacological agents.

Prenatal N-acetylcysteine prevents cigarette smoke-induced lung cancer in neonatal mice.

Certain adult diseases may have their origin early in life, and perinatal exposures may contribute to cancers both during childhood and later in life. We recently demonstrated that mainstream cigarette smoke (MCS) induces a potent carcinogenic response in mice when exposure starts soon after birth. We also showed that the antioxidant N-acetylcysteine (NAC) prevents the extensive nucleotide and gene expression alterations that occur 'physiologically' at birth in mouse lung. The present study was designed to evaluate whether administration of NAC during pregnancy may affect the yield of tumors in mice exposed to MCS, starting after birth and continuing for 120 days. The results obtained showed that 210 days after birth, one adenoma only was detectable in sham-exposed mice. In contrast, as much as the 61.1% (33/54) of MCS-exposed mice born from untreated dams had lung tumors, including both benign tumors and bronchoalveolar carcinomas. Treatment with NAC during pregnancy strikingly inhibited the formation of benign lung tumors and totally prevented occurrence of carcinomas. In addition, prenatal NAC inhibited the MCS-induced hyperplasia of the urinary bladder epithelium. These findings demonstrate for the first time that treatment during pregnancy with an antioxidant chemopreventive agent can affect the induction of tumors consequent to exposure to a carcinogen after birth.

Preneoplastic and neoplastic lesions in the lung, liver and urinary tract of mice exposed to environmental cigarette smoke and UV light since birth.

It is difficult to reproduce the carcinogenicity of cigarette smoke (CS) in animal models. Recently, we showed that exposure of mice to mainstream CS (MCS) for 120 days, starting immediately after birth, resulted in an early and potent carcinogenic response. In parallel, we implemented studies evaluating intermediate biomarkers and tumors in mice exposed to environmental CS (ECS). To this purpose, we used 263 newborn CD-1 mice born from 27 dams. The whole-body exposure to ECS for 120 days, starting within 12 hr after birth, resulted in an early appearance of preneoplastic lesions in lung, which however tended to attenuate after discontinuing exposure. When the experiment was stopped, after 330 days, the number of lung adenomas was higher in ECS-exposed mice as compared to sham-exposed mice, but such increase was statistically significant only in mice co-exposed to smoke and halogen light mimicking solar irradiation. Moreover, exposure to ECS produced extensive histopathological changes, mainly parenchymatous degeneration, in liver. The alterations produced in both lung and liver require that exposure to ECS starts immediately after birth, no effect being observed when exposure started 8 days later. In contrast, induction by ECS of alterations in the urinary tract, such as microadenomas and adenomas in renal pelvis and kidney, papillary hyperplasia of urothelium, and urinary bladder papillomas, were unrelated to the exposure time after birth. The results obtained with ECS cannot be directly compared to those previously obtained with MCS, since the latter involved shorter daily exposures to more massive CS doses.

Potent carcinogenicity of cigarette smoke in mice exposed early in life.

In spite of the dominant role of cigarette smoke (CS) in cancer epidemiology, all studies performed during the past 60 years have shown that this complex mixture is either negative or weakly tumorigenic in experimental animals. We implemented studies aimed at evaluating whether exposure of mice early in life may enhance susceptibility to CS carcinogenicity. A total of 98 newborn Swiss albino mice were either untreated (controls) or received a subcutaneous injection of benzo(a)pyrene [B(a)P] (positive control) or were exposed whole-body to mainstream cigarette smoke (MCS) for 120 days, starting within 12 h after birth. Complete necropsy and histopathological analyses were performed at periodical intervals. In contrast with the lack of lung tumors in controls, MCS-exposed mice developed microscopically detectable tumors, starting only 75 days after birth and reaching an overall incidence of 78.3% after 181-230 days. The mean lung tumor multiplicities were 6.1 and 13.6 tumors per mouse in males and females, respectively, showing a significant intergender difference. Most tumors were microadenomas or adenomas, but 18.4% of the mice additionally had malignant lung cancer. MCS also induced bronchial and alveolar epithelial hyperplasia, and blood vessel proliferation. Furthermore, malignant tumors, some of which may have a metastatic origin, were detected in the urinary tract and liver of MCS-exposed mice. A somewhat different spectrum of tumors was observed in B(a)P-treated mice. In conclusion, MCS is a potent and broad spectrum carcinogen in mice when exposure starts early in life, covering stages of life corresponding to neonatal, childhood and adolescence periods in humans. This animal model will be useful to explore the mechanisms involved in CS-induced carcinogenesis and to investigate the protective effects of dietary agents and chemopreventive drugs.